Intra- and Interspecific Variation of the Mitochondrial Genome in RATTUS NORVEGICUS and RATTUS RATTUS: Restriction Enzyme Analysis of Variant Mitochondrial DNA Molecules and Their Evolutionary Relationships

Restriction endonuclease analysis has revealed extensive mtDNA polymorphism in two species of rats, Rattus rattus and Rattus norvegicus. Sequence divergence values for the eight detected R. norvegicus variants range from 0.2% to 1.8% and for the eight R. rattus variants, from 0.2% to 9.6%. Three of the most closely related R. norvegicus mtDNA's appear to differ by deletions/insertions of about 4 base pairs apiece. Restriction sites for seven enzymes have been mapped for 11 of these variants. The 31 intraspecific and 41 interspecific variant sites appear to be evenly distributed on the mtDNA molecule outside of the rRNA cistrons. The location of sites present in all the DNAs suggests that the rRNA genes and possibly the light strand origin of replication may be more highly evolutionarily conserved than other parts of the molecule. The sequence divergences among the mtDNAs of animals whose geographic origins are separated by major barriers, such as oceans, were significantly greater than those among animals found within large land masses, such as the continental United States. Dendrograms (phenograms), which have been constructed to depict the relationships among the various DNAs, indicate that East Asian members of the R. rattus species are more closely related to American rats of this species than to other Asian R. rattus animals from Sri Lanka. Moreover, it appears that R. norvegicus comprises a group taxonomically distinct from any of the R. rattus subspecies.     

Black rat (Rattus rattus) genomic variability characterized by chromosome painting

Black rats are of outstanding interest in parasitology and infective disease analysis. We used chromosome paints from both the mouse(Mus musculus) and the Norway rat(Rattus norvegicus) to characterize the genome of two Black rat subspecies from Italy. Both subspecies have two large metacentrics (n. 1, 4) not present in the Norway rat (2n = 42).Rattus rattus rattus has a diploid number of 2n = 38, whileRattus rattus frugivorous has two small metacentric “supernumerary” or B chromosomes for a diploid number of 2n = 38 + 2B. The 21 mouse paints gave 38 signals on theR. r. rattus karyotype and 39 signals in theR. r. frugivorous karyotype. The two metacentrics, not present inR. norvegicus, were hybridized by mouse 16/1/17 and mouse 4/10/15. These chromosomes are homologous to: RRA1 = RNO 5/7, and RRA4 = RNO 9/11 and not “4/7” and “11/12” as previously reported. Furthermore, the synteny of Chr 13 of theR. r. frugivorous withR. norvegicus Chr 16 and mouse Chrs 8/14 is not complete, because there is a small pericentromeric insertion of RNO Chr 18 (mouse Chr 18). If we consider only the two metacentrics, RRA1 and RRA4, the principal differences betweenR. norvegicus andR. rattus, then we can propose the derived synteny of 124 genes in the black rat. A comparison of the Z index between rats and mice shows an acceleration of genomic evolution among genus, species, and subspecies. The chromosomal differences betweenR. r. rattus xR. r. frugivorous suggest that they may be classified as different species because hybrids would produce 50% unbalanced gametes.     

Geographical Distribution of Twelve Transferrin Alleles in Black Rats of Asia and Oceania

About 450 black rats (Rattus rattus) were collected from 25 localities in Asia and Oceania. Their serum transferrins were analyzed by a newly developed thin layer acrylamide gel electrophoresis accompanied with acrinol pretreatment, exhibiting 12 transferrin bands. Generally, Asian type rats (2N=42) had fast-moving transferrins (R-series), Ceylon type (2N=40) moderately moving ones and Oceanian type (2N=38) slowly moving ones (C-series). Exceptionally, in northern India and Pakistan all Asian-type rats had C-series Tf. The possibility that divergence of R-series Tf and C-series Tf had preceded the karyotypic differentiation from 42 to 38 is proposed. In combination with the previous molecular data, the time of the divergence is roughly estimated between the order of a million years and ten thousand years.     

Cryptic genetic diversity in Rattus of the San Francisco Bay region, California

Invasive species can have complex invasion histories, harbor cryptic levels of diversity, and pose taxonomic problems for pest management authorities. Roof rats, Rattus rattus sensu lato, are common invasive pests of the San Francisco Bay Area in California, USA. They are a significant health risk and pest management efforts impose a large financial investment from public institutions and private individuals. Recent molecular genetic and taxonomic studies of black rats in their native range in Asia have shown that the species is a complex of two karyotypic forms and four mitochondrial genetic lineages that may represent four distinct species. We used mtDNA sequences and nuclear microsatellite variation to identify which mitochondrial lineages of the R. rattus group are present in the San Francisco Bay Area and to test for gene flow among them. We recovered specimens with mtDNA sequences representing two of the major mtDNA lineages of the R. rattus group. Microsatellite variation, however, was not structured in concordance with mtDNA lineages, suggesting a more complex history involving hybridization and introgression between these lineages. Although Aplin et al. (2011) and Lack et al. (2012) reported R. rattus Lineage II in North America, this is the first detailed examination of possible gene flow amongst lineages in this region.     

Biochemical differentiation among karyotypic forms of Australian Rattus

Isozyme electrophoresis of up to 55 loci, and microcomplement fixation of albumin were used to assess the extent of structural gene divergence among karyotypic forms of Australian Rattus. The results show that the Australian Rattus are monophyletic with respect to R. rattus or R. norvegicus. Within the Australian Rattus, rates of chromosomal evolution have varied enormously, the highest rates being found among members of the R. sordidus group, where extensive chromosomal repatterning has occurred with little or no structural gene divergence.     

Sexual-size dimorphism in two synanthropic rat species: Comparison and eco-evolutionary perspectives

Sexual dimorphism is a common trait in many mammal species and sexual-size dimorphism (SSD) represents its commonest form. Rattus rattus and Rattus norvegicus are two cosmopolitan, polygynous species, for which a male-biased SSD has been anecdotally reported, but never quantified. In this work, we assessed the occurrence of SSD in both species and we tested the hypothesis that R. norvegicus has a more evident SSD than R. rattus, in agreement with their body mass-testes size ratio, intra-male aggressive behaviour and mating system. We collected weight data of 40 (20 males and 20 females) adult R. rattus and 27 (13 males and 14 females) adult R. norvegicus from 4 localities in Italy characterized by different habitat typologies. We used a t-test based on Bayesian inference to compare the SSD in both species. The results were in line with our expectation supporting a higher SSD in R. norvegicus than in R. rattus. This study aimed to identify the eco-evolutionary drivers of SSD, and provides further support to well established life history theories on two widely distributed rodent species.     

Comparative phylogeography of invasive Rattus rattus and Rattus norvegicus in the U.S. reveals distinct colonization histories and dispersal

Invasive Rattus are arguably the most costly and destructive invasive species on the planet, but little is known concerning their invasion history and population structure in the U.S. We utilized both nuclear microsatellites and mitochondrial DNA sequences (mtDNA) to compare the colonization history, patterns of gene flow, and levels of genetic diversity of Rattus rattus and R. norvegicus in the U.S. Analyses of mtDNA suggest R. rattus is characterized by a single rapid expansion into the U.S. from one or two very closely related mtDNA lineages or geographic sources. For R. norvegicus, mtDNA analyses suggest at least four invasions distinct in space and/or time have occurred to establish its distribution in the U.S. Microsatellite analyses suggest for R. rattus that dispersal is characterized by an isolation-by-distance pattern, suggesting a relatively low frequency of long distance dispersal, and low levels of establishment for novel propagules. In contrast, microsatellite analyses of R. norvegicus suggest high frequencies of long distance dispersal and essentially panmixia among nearly all sampled populations, as well as a high frequency of novel propagules entering at the east and west coasts and assimilating into established populations. We discuss these results in the context of invasive Rattus management in the U.S. and their implications for invasive species in general, as well as the implications for managing the spread of rat-borne pathogens.     

Genetics of the ceramide/sphingosine-1-phosphate rheostat in blood pressure regulation and hypertension

Background

South Africa's long and extensive trade activity has ensured ample opportunities for exotic species introduction. Whereas the rich biodiversity of endemic southern African fauna has been the focus of many studies, invasive vertebrates are generally overlooked despite potential impacts on biodiversity, health and agriculture. Genetic monitoring of commensal rodents in South Africa which uncovered the presence of Rattus tanezumi, a South-East Asian endemic not previously known to occur in Africa, provided the impetus for expanded studies on all invasive Rattus species present.

Results

To this end, intensified sampling at 28 South African localities and at one site in Swaziland, identified 149 Rattus specimens. Cytochrome b gene sequencing revealed the presence of two R. tanezumi, seven Rattus rattus and five Rattus norvegicus haplotypes in south Africa. Phylogenetic results were consistent with a single, recent R. tanezumi introduction and indicated that R. norvegicus and R. rattus probably became established following at least two and three independent introductions, respectively. Intra- and inter-specific diversity was highest in informal human settlements, with all three species occurring at a single metropolitan township site. Rattus norvegicus and R. rattus each occurred sympatrically with Rattus tanezumi at one and five sites, respectively. Karyotyping of selected R. rattus and R. tanezumi individuals identified diploid numbers consistent with those reported previously for these cryptic species. Ordination of bioclimatic variables and MaxEnt ecological niche modelling confirmed that the bioclimatic niche occupied by R. tanezumi in south Africa was distinct from that occupied in its naturalised range in south-east Asia suggesting that factors other than climate may influence the distribution of this species.

Conclusions

This study has highlighted the value of genetic typing for detecting cryptic invasive species, providing historical insights into introductions and for directing future sampling. The apparent ease with which a cryptic species can become established signals the need for broader implementation of genetic monitoring programmes. In addition to providing baseline data and potentially identifying high-risk introduction routes, the predictive power of ecological niche modelling is enhanced when species records are genetically verified.     

Commensalism facilitates gene flow in mountains: a comparison between two Rattus species

Small mammal dispersal is strongly affected by geographical barriers. However, commensal small mammals may be passively transported over large distances and strong barriers by humans (often with agricultural products). This pattern should be especially apparent in topographically complex landscapes, such as mountain ranges, where valleys and/or peaks can limit dispersal of less vagile species. We predict that commensal species would have lower genetic differentiation and higher migration rates than related non-commensals in such landscapes. We contrasted population genetic differentiation in two sympatric Rattus species (R. satarae and R. rattus) in the Western Ghats mountains in southern India. We sampled rats from villages and adjacent forests in seven locations (20–640 km apart). Capture-based statistics confirmed that R. rattus is abundant in human settlements in this region, whereas R. satarae is non-commensal and found mostly in forests. Population structure analyses using ~970-bp mitochondrial control region and 17 microsatellite loci revealed higher differentiation for the non-commensal species (R. satarae F-statistics=0.420, 0.065, R. rattus F-statistics=0.195, 0.034; mitochondrial DNA, microsatellites, respectively). Genetic clustering analyses confirm that clusters in R. satarae are more distinct and less admixed than those in R. rattus. R. satarae shows higher slope for isolation-by-distance compared with R. rattus. Although mode of migration estimates do not strongly suggest higher rates in R. rattus than in R. satarae, they indicate that migration over long distances could still be higher in R. rattus. We suggest that association with humans could drive the observed pattern of differentiation in the commensal R. rattus, consequently impacting not only their dispersal abilities, but also their evolutionary trajectories.     

Genetic differentiation in Indonesian and French rats of the subgenus Rattus

The allozyme polymorphism at 28 loci was examined in four Indonesian species of Rattus (R. diardii, R. tiomanicus, R. argentiventer and R. exulans) and in two French species (R. rattus and R. norvegicus). R. diardii was the most polymorphic species (H = 0.089) and R. tiomanicus and R. exulans the least polymorphic (H = 0.026). The six species were found to differ from one another by 2–7 diagnostic loci. The use of allozyme characters for systematic identification is discussed. Genetic relationships between the six species were established using two different methods. In both cases, the results were different from those published earlier. These results are discussed in view of the representatives of the loci investigated.     

Prevalence and Genetic Properties of Salmonella enterica Serovar Typhimurium Definitive Phage Type 104 Isolated from Rattus norvegicus and Rattus rattus House Rats in Yokohama City, Japan

Salmonella enterica serovar Typhimurium was isolated from the intestinal contents of Rattus rattus and Rattus norvegicus house rats captured at two buildings, designated buildings J and YS, in Yokohama City, Japan. From October 1997 to September 1998, 52 of 339 (15.3%) house rats were found to carry Salmonella serovar Typhimurium definitive phage type 104 (DT104). In building J, 26 of 161 (16.1%) house rats carried DT104 over the 1-year study period, compared to 26 of 178 (14.6%) rats in building YS. The isolation rates of DT104 from R. rattus and R. norvegicus were similar in the two buildings. Most DT104 strains from building J (24 of 26) showed resistance to ampicillin, chloramphenicol, streptomycin, sulfisoxazole, and tetracycline and contained both the 1.0- and 1.2-kbp integrons, carrying genes pse1, pasppflo-like, aadA2, sulI, and tet(G). All DT104 strains from building YS were resistant to ampicillin and sulfisoxazole, and had the 1.2-kbp integron carrying pse1 and sulI. Cluster analysis of pulsed-field gel electrophoresis patterns of BlnI-digested DT104 DNAs showed that 22 of 26 DT104 strains from building J and 24 of 26 strains from building YS could be grouped into separate clusters each specific for the building origin. These results indicated that DT104 strains were prevalent in house rat colonies in each building and suggest that house rats may play an important role in the epidemiology of DT104.     

GEOTROPIC ORIENTATION OF YOUNG RATS

The geotropic orientation of Rattus rattus (roof rat) obeys the equations previously found applicable for Rattus norvegicus. The former is more sensitive, geotiopically, and the numerical values of the constants in the equations for the two forms are found to differ significantly. Certain consequences of this difference are pointed out.     

Chromosome evolution in Australian rodents

A total of 219 wild caught specimens representing 12 of the currently recognised 13 species and subspecies of Australian Rattus have been karyotyped. No two species possessed karyotypes in common, most species and several subspecies differing markedly in chromosome number. While the diploid number varied from 2n=32 to 2n=50, the fundamental number (FN) varied only from 60 to 62, suggesting that Robertsonian rearrangements have played a major role in karyotypic evolution in the group. — Karyotypically the Australian species of Rattus fall into two groups. — the R. lutreolus group and the R. sordidus group. Of the karyotypic forms encountered in the former group, that of R. lutreolus is probably most ancestral because it is identical to that of many Asian species of Rattus. Other karyotypic forms in the R. lutreolus group can be derived as follows: That of (1) R. tunneyi tunneyi and R. t. culmorum by a single fixed pericentric inversion; (2) R. fuscipes fuscipes, R. f. greyi, R. f. assimilis and R. f. coracius by two fixed fusions; (3) R. leucopus cooktownensis by three fixed fusions; and (4) R. leucopus leucopus by four fixed fusions. Of the R. sordidus group, R. s. villosissimus may possess the most ancestral karyotype with 2n=50 (FN=60), from which R. s. colletti (2n=42; FN=60) is derived by four fusions and R. s. sordidus (2n=32; FN=60) by nine fusions, four of which appear to be homologous with those R. s. colletti. — The karyotypic data are in accord with Taylor and Horner's (1973) suggestions that (1) R. t. tunneyi and R. t. culmorum belong to one species; (2) R. lut. lutreolus and R. lut. velutinus belong to one species; (3) R. leu. leucopus and R. leu. cooktownensis belong to one species and (4) R. f. fuscipes, R. f. greyi, R. f. assimilis and R. f. coracius belong to one species. However, the large karyotypic difference between R. s. sordidus and R. s. colletti and R. s. villosissimus may indicate that these groups belong to different biological species. — Supernumerary or B-chromosomes were found in R. f. assimilis and R. t. tunneyi. A single R. t. culmorum was heterozygous for a centric fusion.     

Genetic characterization of black rat (Rattus rattus) of the Canary Islands: origin and colonization

In the Canary Islands two invasive rat species, Rattus rattus and Rattus norvegicus are present, but little is known about the origin and colonization. To this end, a molecular study was performed on R. rattus from the Archipelago and from the nearest continents. Partial cytochrome b gene sequencing offered very low levels of haplotype and nucleotide diversities, with only seven haplotypes identified. All of them belong to the European Lineage I, specifically to the “ship rat” cluster. The haplotype network showed a star-like topology. Haplotype distribution showed a genetic subdivision between eastern and central/western islands, suggesting a double colonization event. This hypothesis is congruent with historical human colonization and it is similar to that proposed for the rodent parasite Hymenolepis diminuta. In addition, a possible role of the Canary Islands as a faunal link with the European and American continents is discussed.     

The Toll→NFκB Signaling Pathway Mediates the Neuropathological Effects of the Human Alzheimer's Aβ42 Polypeptide in Drosophila

Alzheimer''s (AD) is a progressive neurodegenerative disease that afflicts a significant fraction of older individuals. Although a proteolytic product of the Amyloid precursor protein, the Αβ42 polypeptide, has been directly implicated in the disease, the genes and biological pathways that are deployed during the process of Αβ42 induced neurodegeneration are not well understood and remain controversial. To identify genes and pathways that mediated Αβ42 induced neurodegeneration we took advantage of a Drosophila model for AD disease in which ectopically expressed human Αβ42 polypeptide induces cell death and tissue degeneration in the compound eye. One of the genes identified in our genetic screen is Toll (Tl). It encodes the receptor for the highly conserved Tl→NFkB innate immunity/inflammatory pathway and is a fly homolog of the mammalian Interleukin-1 (Ilk-1) receptor. We found that Tl loss-of-function mutations dominantly suppress the neuropathological effects of the Αβ42 polypeptide while gain-of-function mutations that increase receptor activity dominantly enhance them. Furthermore, we present evidence demonstrating that Tl and key downstream components of the innate immunity/inflammatory pathway play a central role in mediating the neuropathological activities of Αβ42. We show that the deleterious effects of Αβ42 can be suppressed by genetic manipulations of the Tl→NFkB pathway that downregulate signal transduction. Conversely, manipulations that upregulate signal transduction exacerbate the deleterious effects of Aβ42. Since postmortem studies have shown that the Ilk-1→NFkB innate immunity pathway is substantially upregulated in the brains of AD patients, the demonstration that the Tl→NFkB signaling actively promotes the process of Αβ42 induced cell death and tissue degeneration in flies points to possible therapeutic targets and strategies.     

Commensal Rats and Humans: Integrating Rodent Phylogeography and Zooarchaeology to Highlight Connections between Human Societies

Phylogeography and zooarchaeology are largely separate disciplines, yet each interrogates relationships between humans and commensal species. Knowledge gained about human history from studies of four commensal rats (Rattus rattus, R. tanezumi, R. exulans, and R. norvegicus) is outlined, and open questions about their spread alongside humans are identified. Limitations of phylogeographic and zooarchaeological studies are highlighted, then how integration would increase understanding of species’ demographic histories and resultant inferences about human societies is discussed. How rat expansions have informed the understanding of human migration, urban settlements, trade networks, and intra- and interspecific competition is reviewed. Since each rat species is associated with different human societies, they identify unique ecological and historical/cultural conditions that influenced their expansion. Finally, priority research areas including nuclear genome based phylogeographies are identified using archaeological evidence to understand R. norvegicus expansion across China, multi-wave colonization of R. rattus across Europe, and competition between R. rattus and R. norvegicus.     

Chromosomal diversity in three species of electric fish (Apteronotidae,Gymnotiformes) from the Amazon Basin

Cytogenetic studies were carried out on samples of Parapteronotus hasemani, Sternarchogiton preto and Sternarchorhamphus muelleri (Apteronotidae, Gymnotiformes) from the Amazon basin. The first two species exhibited both a 2n = 52 karyotype, but differed in their karyotypic formulae, distribution of constitutive heterochromatin, and chromosomal location of the NOR. The third species, Sternarchorhamphus muelleri, was found to have a 2n = 32 karyotype. In all three species the DAPI and chromomycin A3 staining results were consistent with the C-banding results and nucleolar organizer region (NOR) localization. The 18S rDNA probe confirmed that there was only one pair of ribosomal DNA cistron bearers per species. The telomeric probe did not reveal interstitial telomeric sequences (ITS). The karyotypic differences among these species can be used for taxonomic identification. These data will be useful in future studies of these fishes and help understanding the phylogenetic relationships and chromosomal evolution of the Apteronotidae.     

High-Throughput Mutation Profiling Changes before and 3 Weeks after Chemotherapy in Newly Diagnosed Breast Cancer Patients

Background

Changes in tumor DNA mutation status during chemotherapy can provide insights into tumor biology and drug resistance. The purpose of this study is to analyse the presence or absence of mutations in cancer-related genes using baseline breast tumor samples and those obtained after exposure to one cycle of chemotherapy to determine if any differences exist, and to correlate these differences with clinical and pathological features.

Methods

Paired breast tumor core biopsies obtained pre- and post-first cycle doxorubicin (n = 18) or docetaxel (n = 22) in treatment-naïve breast cancer patients were analysed for 238 mutations in 19 cancer-related genes by the Sequenom Oncocarta assay.

Results

Median age of patients was 48 years (range 32–64); 55% had estrogen receptor-positive tumors, and 60% had tumor reduction ≥25% after cycle 1. Mutations were detected in 10/40 (25%) pre-treatment and 11/40 (28%) post-treatment samples. Four mutation pattern categories were identified based on tumor mutation status pre- → post-treatment: wildtype (WT)→WT, n = 24; mutant (MT)→MT, n = 5; MT→WT, n = 5; WT→MT, n = 6. Overall, the majority of tumors were WT at baseline (30/40, 75%), of which 6/30 (20%) acquired new mutations after chemotherapy. Pre-treatment mutations were predominantly in PIK3CA (8/10, 80%), while post-treatment mutations were distributed in PIK3CA, EGFR, PDGFRA, ABL1 and MET. All 6 WT→MT cases were treated with docetaxel. Higher mutant allele frequency in baseline MT tumors (n = 10; PIK3CA mutations n = 8) correlated with less tumor reduction after cycle 1 chemotherapy (R = -0.667, p = 0.035). No other associations were observed between mutation pattern category with treatment, clinicopathological features, and tumor response or survival.

Conclusion

Tumor mutational profiles can change as quickly as after one cycle of chemotherapy in breast cancer. Understanding of these changes can provide insights on potential therapeutic options in residual resistant tumors.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00212082","term_id":"NCT00212082"}}NCT00212082     

Evolution of karyotypes and differentiation in 13 Rattus Species

Karyotypes of 13 Rattus species collected in Asia and Oceania were analysed with special interest to karyotype evolution and species differentiation. They were classified into three groups according to their karyotype similarity. Four species (R. annandalei, R. exulans, R. muelleri and R. norvegicus) with 2n=42 and a karyotype similar to some of the polymorphic karyotypes in the Asian black rats (R. rattus) are classified into the first group. Pericentric inversion of some acrocentrics seemed to have caused the differentiation of these species. The other four species (R. bowersii, R. fuscipes, R. leucopus and R. conatus) with similar karyotypes as the above group, but lower chromosome numbers than 2n=42 are classified into the second group. Robertsonian fusion in some acrocentrics observed in the first group are suggested to have caused the development of the species in this group. The remaining four species (R. sabanus, R. canus, R. huang and R. niviventer) with karyotypes markedly different from the above two in having a fewer number of small metacentrics are classified into the third group. They seemed to be more primitive karyotypes than the other Rattus species. By the comparison between the polymorphic karyotypes in the black rat, and karyotypes in its related species it was suggested that the former had occurred as primary events to the differentiation of the latter. Parallelism between the karyotype evolution and the species differentiation was discussed.Contribution No. 874 from the National Institute of Genetics, Japan. Supported by a grant-in-aid from the Ministry of Education of Japan (No. 92159 and 92332).     

Alzheimer Presenilin-1 Mutations Dramatically Reduce Trimming of Long Amyloid β-Peptides (Aβ) by γ-Secretase to Increase 42-to-40-Residue Aβ

The presenilin-containing γ-secretase complex produces the amyloid β-peptide (Aβ) through intramembrane proteolysis, and >100 presenilin mutations are associated with familial early-onset Alzheimer disease (AD). The question of whether these mutations result in AD through a gain or a loss of function remains highly controversial. Mutations in presenilins increase ratios of 42- to 40-residue Aβ critical to pathogenesis, but other Aβs of 38–49 residues are also formed by γ-secretase. Evidence in cells suggests the protease first cleaves substrate within the transmembrane domain at the ϵ site to form 48- or 49-residue Aβ. Subsequent cleavage almost every three residues from the C terminus is thought to occur along two pathways toward shorter secreted forms of Aβ: Aβ49 → Aβ46 → Aβ43 → Aβ40 and Aβ48 → Aβ45 → Aβ42 → Aβ38. Here we show that the addition of synthetic long Aβ peptides (Aβ45–49) directly into purified preparations of γ-secretase leads to the formation of Aβ40 and Aβ42 whether the protease complex is detergent-solubilized or reconstituted into lipid vesicles, and the ratios of products Aβ42 to Aβ40 follow a pattern consistent with the dual-pathway hypothesis. Kinetic analysis of five different AD-causing mutations in presenilin-1 revealed that all result in drastic reduction of normal carboxypeptidase function. Altered trimming of long Aβ peptides to Aβ40 and Aβ42 by mutant proteases occurs at multiple levels, independent of the effects on initial endoproteolysis at the ϵ site, all conspiring to increase the critical Aβ42/Aβ40 ratio implicated in AD pathogenesis. Taken together, these results suggest that specific reduction of carboxypeptidase function of γ-secretase leads to the gain of toxic Aβ42/Aβ40.